The liver is the largest organ in the body. It is involved in a myriad of metabolic processes required for body homeostasis and can also detoxify harmful chemicals. The liver receives blood from the hepatic portal vein and the hepatic artery. The hepatic portal vein carries blood from the intestines, pancreas, spleen and gallbladder and exposes the liver to nutrients and toxins derived from these sources. An unfortunate result of the liver's exposure to ingested substances is that some of these substances, such as alcohol, are harmful to the liver. The liver is also a target for persistent viral infections, such as chronic infections with the hepatitis B virus (HBV). Epidemiological studies have linked some types of infectious agents and alcohol consumption with liver diseases and liver cancer;however, attempts to fully characterize many of these diseases in humans have been hampered by the absence of experimentally tractable human liver model systems. Impediments that have limited development of human liver model systems include the difficulty in maintaining differentiated hepatocytes in traditional cell-culture environments and the difficulty in obtaining large numbers of normal human hepatocytes. Hepatocytes are the predominant cell type in the liver and are responsible for many activities that are attributed to the liver;normal hepatocyte function is maintained by communication and contact with other liver cells such as liver sinusoidal endothelial cells (LSEC). The heterogeneity in metabolic processes throughout the liver has generated debate over what defines a functional liver unit. However, even considering this heterogeneity, the liver sinusoid and surrounding cells can be viewed as the most basic structural and functional unit of the liver. This proposal is directed towards: 1.) Developing a novel "mini-liver" culture system using a microfluidic platform that simulates a liver sinusoid functional unit, and 2.) Testing the utility of this system with two focused studies that analyze the effect of hepatitis B virus (HBV) replication and alcohol exposure on specific hepatocyte signaling pathways. The microfluidic platform will have a layered co-culture of hepatocytes and LSECs in a microchannel that mimics a liver sinusoid functional unit. We will initially develop a simple hepatocyte/collagen/LSEC sandwiched configuration and then expand this to include a channel for bile removal and, if necessary, additional sinusoid resident cells such as Kupffer or stellate cells. Exchange of fluid will be regulated through microchannels and external flow control units. We hypothesize that this system will generate an authentic, simple liver sinusoid functional unit. The current proposal focuses on establishing this system and studying the impact of HBV infections and alcohol exposure on hepatocyte physiology. Our long-term goal is to develop a novel human liver model system that can be used to study normal human liver biology. Future use of this system could include studies to understand the impact of other infectious agents on the liver, the consequence of liver exposure to toxins and the effects of various oxygen concentrations on the known heterogeneity of hepatocyte metabolism. PUBLIC HEALTH RELEVANCE: Our understanding of how various human liver diseases develop, such as those associated with persistent viral infections or exposure to toxins or alcohol, has been hampered by the difficulty of acquiring large numbers of human hepatocytes and maintaining their liver-specific functions when grown in traditional cell-culture systems. We propose to use recent advances in bioreactor and microfluidic platform technology to create a novel human liver model system in which small number of human liver cells will be cultured in environmentally controlled microchannels to create a functional liver unit and a novel model for studying human liver functions and diseases.